CN113075410B - Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon - Google Patents

Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon Download PDF

Info

Publication number
CN113075410B
CN113075410B CN202110333807.7A CN202110333807A CN113075410B CN 113075410 B CN113075410 B CN 113075410B CN 202110333807 A CN202110333807 A CN 202110333807A CN 113075410 B CN113075410 B CN 113075410B
Authority
CN
China
Prior art keywords
nrnp
antibody
diagnosis
quantitative detection
detection agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110333807.7A
Other languages
Chinese (zh)
Other versions
CN113075410A (en
Inventor
张彦
朱云
曾纪晓
夏慧敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Women and Childrens Medical Center
Original Assignee
Guangzhou Women and Childrens Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Women and Childrens Medical Center filed Critical Guangzhou Women and Childrens Medical Center
Priority to CN202110333807.7A priority Critical patent/CN113075410B/en
Publication of CN113075410A publication Critical patent/CN113075410A/en
Priority to PCT/CN2022/082966 priority patent/WO2022206585A1/en
Application granted granted Critical
Publication of CN113075410B publication Critical patent/CN113075410B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of biomedicine, in particular to application of an anti-nRNP/Sm antibody as a diagnosis marker of Hirschsprung's disease. The invention has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of the congenital diseases of children, and fills the blank of the diagnosis of the congenital megacolon plasma. The method has good diagnosis sensitivity and specificity, the AUC value is 0.8165, the optimal limit corresponds to the sensitivity of 100 percent, the specificity is 64.10 percent, and an effective method is provided for the diagnosis of the congenital megacolon.

Description

Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon
Technical Field
The invention relates to the field of biomedicine, in particular to application of an anti-nRNP/Sm antibody as a diagnosis marker of Hirschsprung's disease.
Background
Hirschsprung disease (HSCR) is a birth defect disease of children with intestinal nerve dysplasia, and the pathological mechanism is that migration and differentiation of intestinal neural crest cells are blocked in intestinal neurons, so that the intestinal nerve is deficient and persistent spasm occurs, and the disease is one of common congenital intestinal tract diseases of children. Early congenital megacolon is manifested as vomiting, abdominal distension, diarrhea and the like, which can lead to death of newborn infants or complications such as repeated enteritis after operation, intractable constipation and the like clinically, and seriously affect the growth, development and life quality of children patients.
The timely diagnosis and treatment of the congenital megacolon can reduce the risk of the congenital megacolon enteritis and obtain good prognosis. The diagnosis of the disease requires pathological sections of the diseased tissue after surgery. The preoperative diagnosis method mainly comprises barium enema, rectal biopsy and rectal manometry to judge whether to implement 'giant colon radical operation'. At present, barium enema is the most important diagnostic method, and the principle is that no nerve segment stenosis and proximal dilatation exist in the intestinal tract of a child suffering from congenital megacolon, and the colon is diagnosed as megacolon by the dilation and the stenosis after barium enema. However, the method can only diagnose the children with typical intestinal tract shape change, the sensitivity needs to be improved, and the diagnosis accuracy is about 80%. Rectal biopsy is to directly take the tissues of the straight intestine and detect whether the ganglion cells are lost, so the accuracy is high, but the sampling part has influence on the result. The method is invasive and very expensive, and is generally not readily applicable to infants who have a barium enema which is not obvious or is not suitable, for example, when Necrotizing Enterocolitis (NEC) is a possibility, the barium enema may cause intestinal perforation, and the barium enema is not suitable for diagnosis, and rectal biopsy is considered. Rectal manometry is to determine the innervation abnormality of the intestinal nerves by detecting the lack of relaxation of the internal anal sphincter, is only an auxiliary diagnosis method, has more false positives and false negatives, and cannot be used for single detection.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an anti-nRNP/Sm antibody of a diagnosis marker of the Hirschsprung's disease and application thereof, and provides a new accurate and sensitive detection way for the diagnosis of the Hirschsprung's disease.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention relates to an application of a quantitative detection agent of an anti-nRNP/Sm antibody in preparation of a diagnostic reagent, a test strip or a kit for Hirschsprung's disease.
Alternatively, for use as described above, the anti-nRNP/Sm antibody is an IgG antibody.
Optionally, for use as described above, the quantitative detection agent is used to perform any one of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
Optionally, in the above-mentioned application, the quantitative detection agent is an antigenic substance selected from the group consisting of nRNP, Sm protein, and a complex of Sm and RNP.
Alternatively, for use as described above, the antigenic material is conjugated to a solid support.
Alternatively, the solid support is selected from the group consisting of test tubes, EP tubes, multi-well plates, wells of a microplate, and microspheres, for use as described above.
Optionally, for use as described above, the quantitative detection reagent further comprises an anti-human Ig antibody.
Alternatively, for use as described above, the anti-human Ig antibody is an anti-human IgG antibody.
Alternatively, the anti-human Ig antibody is conjugated with a signal agent for use as described above.
Optionally, in the above-mentioned application, the sample to be tested detected by the quantitative detection agent is at least one of a blood, plasma, serum, tissue, cell, tissue or cell lysate sample.
The invention has the beneficial effects that:
the invention has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of the congenital diseases of children, and fills the blank of the diagnosis of the congenital megacolon plasma. The method has good diagnosis sensitivity and specificity, the AUC value is 0.8165, the optimal limit corresponds to the sensitivity of 100 percent, the specificity is 64.10 percent, and an effective method is provided for the diagnosis of the congenital megacolon.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram showing an example of screening for diagnostic markers in plasma of a child having Hirschsprung's disease using a human autoantigen chip according to the present invention;
FIG. 2 is a graph showing the enzyme-linked immunosorbent assay (ELISA) assay for the level of anti-nRNP/Sm antibody in the infant with congenital megacolon and other controls according to one embodiment of the present invention;
FIG. 3 is a graph showing the diagnostic value of the ROC curve analysis of anti-nRNP/Sm antibodies in the congenital megacolon in one embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention relates to application of a quantitative detection agent of an anti-nRNP/Sm antibody in preparation of a diagnostic reagent, a test strip or a kit for Hirschsprung's disease.
The invention discovers for the first time that the anti-nRNP/Sm antibody can be used as a diagnosis marker of the congenital megacolon, has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of the congenital diseases of children, and fills the blank of the diagnosis of the congenital megacolon plasma. The method has good diagnosis sensitivity and specificity, the AUC value is 0.8165, the optimal limit corresponds to the sensitivity of 100 percent, the specificity is 64.10 percent, and an effective method is provided for the diagnosis of the congenital megacolon.
The term "marker" or "biochemical marker" as used herein refers to a molecule to be used as a target for analyzing a patient test sample.
In some embodiments, the anti-nRNP/Sm antibody is an IgG antibody.
In the determination method of the present invention, the method of expression analysis of the autoantibody against the nRNP, Sm protein, and the complex of Sm and RNP is not particularly limited. For example, the absolute amount or concentration of these autoantibodies in a blood sample is not limited to measurement, and a relative amount or concentration may be measured. More specifically, for example, the amount, concentration or activity of the autoantibody or the like in a blood sample can be measured. Such methods are well known in the art, and by way of example, in some embodiments, the quantitative detection agent is used to perform any of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
Examples of the above-mentioned detection method include a time-of-flight MASS spectrometry (TOF-MASS) such as matrix assisted laser desorption/ionization time-of-flight MASS spectrometry (MALDI-TOF-MASS) and surface enhanced laser desorption/ionization time-of-flight MASS spectrometry (SELDI-TOF-MASS). The concentration or amount of autoantibodies can be grasped from the TOF-MASS chart by the molecular weight peak, other fragment peaks, their intensities, and the like. In addition, as in the case of ELISA, autoantibodies that selectively bind to the protein chip can also be detected using a secondary antibody that has a labeling group and can bind to the respective antibodies.
Among these methods, immunoassay is preferable as a method for performing the expression analysis. The immunoassay method has high sensitivity and accuracy, and can detect a slight change in the concentration of autoantibodies in blood. When the expression of the autoantibody according to the present invention is analyzed by enzyme-linked immunosorbent assay (ELISA), the diagnostic kit for colons congenital megacolon according to the present invention can be suitably used.
In some embodiments, the quantitative detection agent is an antigenic substance selected from the group consisting of nRNP, Sm protein, and complexes of Sm and RNP.
An autoantigen fragment comprising an epitope recognized by an autoantibody can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 amino acids in length. The fragment can also be between 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, or 250 and one amino acid less than the full length of the autoantigen. Typically, these epitopes are characterized in advance so that autoantibodies to a given autoantigen are known to recognize the epitope. Methods of epitope mapping are well known in the art. An "epitope" is a site on an antigen, such as an autoantigen disclosed herein, that is recognized by an autoantibody. Obviously, the quantitative detection agent may also be at least one peptide selected from the group consisting of a fragment peptide, a denatured product, and a modified product of an antigenic substance. The denatured product of the antigen substance is obtained by physical treatment such as heating, freezing, or ultraviolet irradiation, or chemical treatment such as application of a surfactant or a denaturant, and is capable of specifically binding to the autoantibody. For example, a denatured product obtained by SDS or DTT treatment is mentioned. The modified product refers to a modified product obtained by modifying 1 or more amino acids and capable of specifically binding to the autoantibody. For example, a modified product obtained by treatment with glutaraldehyde may be mentioned. The above-mentioned peptide may have mutation, substitution, deletion and/or addition of 1 or several amino acid residues as long as it can specifically bind to the above-mentioned autoantibody.
In some embodiments, the antigenic material is conjugated to a solid support.
In some embodiments, the solid support is selected from the group consisting of a test tube, an EP tube, a multi-well plate, a microplate well, and a microsphere.
The term "solid support" means that the carrier material is predominantly non-liquid-resistant, thereby allowing accurate and traceable localization of nucleic acids on the carrier material. The solid support can be selected from polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel, etc. The preferred solid support is an enzyme label plate. It may contain 16, 32, 48, 64, 96 or more holes.
In the present invention, the term "microsphere" may be a sphere, a nearly sphere, a cube, a polyhedron or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400nm to 10 μm.
The microspheres have specific binding properties for the substance of interest (target or analyte) to be assayed on their surface.
The microspheres are preferably magnetic beads, and the magnetic material is contained in the composition. The magnetic substance may be a metal (elemental metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe) 2 O 3 Or Fe 3 O 4 Magnetic nanoparticles); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.
The surface of the microsphere is modified with one or more active functional groups, wherein the active functional groups comprise-OH, -COOH and-NH 2 -CHO, and-SO 3 H. In some embodiments, the coated antigen and antibody are conjugated or bound to the microsphere by physisorption or direct chemical conjugation (e.g., bridging by a bridge). In particular, suitable techniques for constructing the bridge include, for example, covalent attachment, adsorption, non-covalent interactions, or combinations thereof. In some embodiments, direct bridging may be achieved by glutaraldehyde fixation, N-hydroxysuccinimide (NHS) chemistry, or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) NHS chemistry. Suitable means for indirect bridging include, for example, bridging via a peptide, protein, antibody, linker, or a combination thereof. In some embodiments, the indirect bridging to the solid support is via streptavidin and biotin.
In some embodiments, the quantitative detection agent further comprises an anti-human Ig antibody.
In some embodiments, the anti-human Ig antibody is an anti-human IgG antibody.
Anti-human Ig antibodies are antibodies directed against human Ig proteins, and anti-human IgG antibodies are antibodies directed against human IgG proteins.
In some embodiments, the anti-human Ig antibody is conjugated to a signal agent.
In other embodiments, the anti-human Ig antibody is not labeled with a signal substance, and the quantitative detection agent further comprises a second antibody against the anti-human Ig antibody labeled with a signal substance. The proteins and the like that have non-specifically bound to the antigen substance and the like are washed and removed with a buffer solution and the like, and then the secondary antibody is allowed to act thereon. The secondary antibody binds to the autoantibody or the like bound to the peptide or the like. The secondary antibody is detected by a method corresponding to the signal substance.
In the present invention, the antibody used as a quantitative detection agent may be of IgA, IgD, IgG, IgE or IgM isotype or in the form of a single domain, such as a single domain antibody from a camelid. In some embodiments, the antibody used as a quantitative detection agent is an IgG antibody.
The buffer comprises, for example, one or more of the following components: phosphate buffer, NaCl, EDTA, Pluronic F-127, sodium azide, sorbitol, sulfhydryl modified bovine serum or any combination, variant or equivalent thereof.
In the present invention, the signal substance is a substance capable of providing a signal to be detected, and in some embodiments, the signal substance is independently selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme. The following non-limiting section lists these markers:
enzymes which produce a detectable signal, e.g.by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescent, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
Groups with electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltage measurement and resistance.
A detectable group, such as one whose molecular size is sufficient to induce a detectable modification in its physical and/or chemical properties; such detection can be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic spectroscopy and tunneling).
Electron-dense substances, e.g. radioactive molecules (e.g. 32 P， 35 S or 125 I)。
In some embodiments, the signal species is an Acridinium Ester (AE).
Further, acridine chemiluminescent substances include acridinium esters and acridinium sulfonamides.
Further, the acridine chemiluminescent substance includes acridine ester AE-NHS, acridine ester DMAE-NHS, acridine ester Me-DMAE-NHS, acridine ester NSP-DMAE-NHS, acridine salt NSP-SA-NHS, acridine hydrazide NSP-SA-ADH, etc.
In some embodiments, the kit may further comprise a sample pretreatment reagent (e.g., a sample purification enrichment reagent, a lysis solution, etc.), a washing solution (e.g., PBS, etc.), a buffer solution, a color reagent for a signal substance (e.g., horseradish peroxidase, and then ECL), and the like.
When the test strip is prepared, the test strip can comprise a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein the reaction membrane is provided with a detection area and a quality detection area;
the conjugate pad is coated with an antibody against a test sample-derived species as described above, and the antibody against the test sample-derived species is conjugated to a signal substance; the detection zone is conjugated with an antigenic substance or fragment thereof as described above.
Any biological sample containing autoantibodies may be used as the sample to be detected, including, but not limited to, serum, plasma, whole blood, saliva, urine, semen, sweat, tears, and body tissue. In some preferred embodiments, the sample to be tested by the quantitative detection agent is at least one of a blood, plasma, serum, tissue, cell, tissue or cell lysate sample.
As used herein, "tissue or cell lysate" may also be used in common with the terms "lysate", "lysed sample", "tissue or cell extract", and the like, to denote a sample and/or biological sample material comprising lysed tissue or cells, i.e. where the structural integrity of the tissue or cells has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. This process is encompassed by the term "lysis".
The diagnostic kit of the present invention preferably contains a normal control sample and a colons hirsutum control sample. When these samples are attached to the kit, the presence or absence of the congenital megacolon of the subject can be determined more objectively by performing the same experiment on these samples and comparing the measurement values with the results of the test sample.
The concentration or amount of autoantibodies contained in the sample is indirectly obtained by the intensity of color development or the like. The obtained measurement values can be converted into relative or absolute concentrations, amounts, activities, and the like by a calibration curve or the like.
The invention also relates to a method for diagnosing the congenital megacolon, which comprises the following steps:
detecting the level of anti-nRNP/Sm antibody in the test sample, wherein an increased level of anti-nRNP/Sm antibody is indicative of the presence of hirschsprung.
The term "indicative" when used in the context of autoantibodies for use with embodiments of the present invention includes autoantibodies whose presence or absence is determined by embodiments of the present invention as typically present in subjects having Hirschsprung's disease. By "normally present" is meant that the autoantibody is often associated with the congenital megacolon. "frequently relevant" includes a probability of more than 50%, preferably more than 60%, more preferably more than 70%, even more preferably more than 80% and particularly preferably more than 90% or 95%.
An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types. As the skilled artisan will appreciate, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Conversely, biochemical markers can be used to assess, for example, the presence or absence or severity of a disease with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.
In the method of the present invention, the presence or absence of the onset of Hirschsprung's disease is then determined from the expression analysis results obtained. That is, the more severe the onset of the congenital megacolon or the symptoms thereof, the higher the concentration or amount of the autoantibody according to the present invention in blood. Thus, based on the results of expression analysis of the autoantibody according to the present invention, a positive antibody can be determined when the expression level is high, and a negative antibody can be determined when the expression level is low.
In fact, the boundary between positive and negative, i.e., cutoff value, can be varied according to the definition and severity of the Hirschsprung's disease, the method of analysis of the expression of the autoantibodies to which the present invention relates. Therefore, at a stage where there is no general reference, it is necessary for the practitioner of the method of the present invention to determine the expression analysis method and the cutoff value in advance by preliminary experiments or the like and then perform the measurement.
In some embodiments, the subject is a patient below 18, e.g., 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year old. Or in infants, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months of age.
Embodiments of the present invention will be described in detail with reference to examples.
The test results of the invention are all analyzed by statistics, t test is used for evaluating the difference between two groups, p is less than 0.05 for representing statistical significance, and two-sided test is used for all p values. Statistical analysis was performed using R and graphpad8.0 software.
The congenital megacolon disease is one of common congenital intestinal diseases of children, namely, the congenital megacolon disease is clinically divided into a short segment type, a common type, a long segment type and a full colon type according to increasing severity because the colon is lack of ganglion cells to cause continuous spasm of an intestinal canal, excrement is stagnated in the proximal colon, and the proximal colon is thickened and expanded. Short-segment lesions are positioned at the near and middle segments of the rectum and are not more than 6.5cm away from the anal canal; the common lesions were located at the proximal rectum end or distal end of the rectosigmoid colon, about 9cm from the anal canal; long segment lesions extend to the sigmoid or descending colon; the colon-wide lesion reaches the whole colon and the tail end of the ileum within 30cm from the ileocecal valve.
The "ROC curve" of the present invention is a curve of 1-specificity (false positive rate) and sensitivity (true positive rate) changes, reflecting the diagnostic capabilities of the classifiers. A good classifier has a ratio of true positive rate to false positive rate of greater than 1, away from the 45 degree line.
Example 1 plasma and tissue sample Collection and grouping
Plasma samples were divided into the hirschsprung child group (37 cases), the other enteropathy control group (18 cases), the healthy child group (30 cases), the age ranged from 3 months to 3 years, the gender male 3/4 was male, and the age and gender of the disease and control groups were matched. All samples were from the Guangzhou city women's Children medical center, and the healthy children group had blood samples left after physical examination. The blood sampling mode is anticoagulation blood sampling, centrifugal separation of blood plasma and freezing storage of samples.
Example 2 screening of human autoimmune antigen chips and analysis of differentially expressed autoantibodies
Plasma of 5 children with megacolon congenita (HSCR), 5 of each plasma of children with healthy group (HC) and other intestinal Diseases (DC), were screened against a human autoimmune antigen chip provided by cantonese biotechnology limited, guangzhou, which contains lgG detection of over 100 autoimmune antibodies, and the raw data, after subtraction of negative control, were normalized by RLM method to obtain results of inter-group difference analysis using M statistics, and fig. 1 shows a cluster analysis graph of the different autoantibodies, from which it can be seen that the anti-nRNP/Sm antibody was significantly higher in plasma of children with megacolon congenita than in the control group (p 0.003).
Example 3 enzyme-linked immunosorbent assay (ELISA) detection of anti-nRNP/Sm antibodies
To verify the diagnostic effect of the anti-nRNP/Sm antibody on the congenital giant colon, plasma from giant colon patients (37 cases), other intestinal disease controls (including 18 plasma samples of children with anal stenosis and intestinal stenosis fistulization), and healthy child controls (30 cases) was collected, and the level of the anti-nRNP/Sm antibody in the plasma was detected by enzyme-linked immunosorbent assay (ELISA), wherein the detection kit was a human anti-nRNP/Sm antibody (nRNP/Sm-Ab) enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Zhen science and technology, Inc.). The results are shown in FIG. 2, which shows that the anti-nRNP/Sm antibodies in the plasma of megacolon patients are significantly higher than the healthy children control group (p <0.01), not different from the intestinal disease control group, and higher than the intestinal disease and healthy children combined control group (p < 0.01).
Example 4 ROC Curve analysis
FIG. 3 shows the ROC curve used to evaluate the diagnostic effect of anti-nRNP/Sm antibodies on Hirschmus. The AUC value was 0.8165, the best margin corresponds to a sensitivity of 100% and a specificity of 64.10%. Therefore, the anti-nRNP/Sm antibody can effectively diagnose the congenital megacolon.
In conclusion, the invention obtains the anti-nRNP/Sm antibody with high expression in the blood plasma of the infant with congenital megacolon through the screening of the human autoimmune antigen chip. And an enzyme-linked immunosorbent assay (ELISA) method is used in an independent sample to verify that the anti-nRNP/Sm antibody in the megacolon group (HSCR) is obviously higher than that of other disease and health control groups, the anti-nRNP/Sm antibody can effectively diagnose the infant with congenital megacolon, the AUC value is 0.8165, the optimal limit corresponding sensitivity is 100%, and the specificity is 64.10%. The anti-nRNP/Sm antibody can be used as a plasma diagnosis marker of the Hirschsprung's disease, is used for screening and diagnosing the disease, and fills the blank of blood diagnosis of the Hirschsprung's disease.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. The application of the quantitative detection agent of the anti-nRNP/Sm autoantibody in the preparation of a diagnostic reagent, a test strip or a kit for the congenital megacolon.
2. The use of claim 1, wherein the anti-nRNP/Sm autoantibody is an IgG antibody.
3. The use according to claim 1, wherein the quantitative detection agent is used to perform any one of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
4. The use according to claim 1, wherein the quantitative detection agent comprises an antigenic substance selected from the group consisting of nRNP, Sm protein, and a complex of Sm and RNP.
5. The use according to claim 4, wherein the antigenic substance is conjugated to a solid support.
6. The use according to claim 5, wherein the solid support is selected from the group consisting of test tubes, EP tubes, multiwell plates, microplate wells, and microspheres.
7. The use of any one of claims 4 to 6, wherein the quantitative detection agent further comprises an anti-human IgG antibody.
8. The use of claim 7, wherein said anti-human IgG antibody is conjugated to a signal agent.
9. The use according to any one of claims 1 to 6 and 8, wherein the sample to be tested to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cells, tissue lysate sample or cell lysate sample.
CN202110333807.7A 2021-03-29 2021-03-29 Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon Active CN113075410B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110333807.7A CN113075410B (en) 2021-03-29 2021-03-29 Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon
PCT/CN2022/082966 WO2022206585A1 (en) 2021-03-29 2022-03-25 Antibody markers for diagnosing hirschsprung disease and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110333807.7A CN113075410B (en) 2021-03-29 2021-03-29 Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon

Publications (2)

Publication Number Publication Date
CN113075410A CN113075410A (en) 2021-07-06
CN113075410B true CN113075410B (en) 2022-08-16

Family

ID=76611086

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110333807.7A Active CN113075410B (en) 2021-03-29 2021-03-29 Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon

Country Status (1)

Country Link
CN (1) CN113075410B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206585A1 (en) * 2021-03-29 2022-10-06 广州市妇女儿童医疗中心 Antibody markers for diagnosing hirschsprung disease and application thereof
CN115462347A (en) * 2021-08-13 2022-12-13 广州市妇女儿童医疗中心 Application of retinoic acid metabolic pathway in congenital megacolon diseases

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1341621A (en) * 2000-09-05 2002-03-27 上海博德基因开发有限公司 A novel polypeptide-human U2 snRNP accessory factor small subunit 26-24.86 and polynucleotide for coding said polypeptide
CN101512007A (en) * 2005-10-13 2009-08-19 人体基因组科学有限公司 Methods and compositions for use in treatment of patients with autoantibody positive diseases
CN102358900A (en) * 2011-11-09 2012-02-22 南京医科大学 Plasma micro-ribonucleic acid (miRNA) marker related with human Hirschsprung's disease and application of miRNA marker
CN103278579A (en) * 2013-05-22 2013-09-04 夏彦恺 Plasma metabolism micromolecular marker related to human intestinal canal aganglionosis and application of plasma metabolism micromolecular marker
CN104459100A (en) * 2014-11-25 2015-03-25 成都威尔诺生物科技有限公司 Anti-SM-RNP antibody kit

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050055733A1 (en) * 2001-07-06 2005-03-10 Zairen Sun Small intestine and colon genes
DE602005022924D1 (en) * 2004-12-08 2010-09-23 Cedars Sinai Medical Ct Los An PROCESS FOR THE DIAGNOSIS OF MORBUS CROHN

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1341621A (en) * 2000-09-05 2002-03-27 上海博德基因开发有限公司 A novel polypeptide-human U2 snRNP accessory factor small subunit 26-24.86 and polynucleotide for coding said polypeptide
CN101512007A (en) * 2005-10-13 2009-08-19 人体基因组科学有限公司 Methods and compositions for use in treatment of patients with autoantibody positive diseases
CN102358900A (en) * 2011-11-09 2012-02-22 南京医科大学 Plasma micro-ribonucleic acid (miRNA) marker related with human Hirschsprung's disease and application of miRNA marker
CN103278579A (en) * 2013-05-22 2013-09-04 夏彦恺 Plasma metabolism micromolecular marker related to human intestinal canal aganglionosis and application of plasma metabolism micromolecular marker
CN104459100A (en) * 2014-11-25 2015-03-25 成都威尔诺生物科技有限公司 Anti-SM-RNP antibody kit

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
儿童系统性红斑狼疮并发假性肠梗阻8例临床研究;唐晓艳，等;《中国实用儿科杂志》;20210131;第36卷(第1期);38-41 *
先天性巨结肠患儿肠道内Smoothelin的表达;沈涤华，等;《中华小儿外科杂志》;20170430;第38卷(第4期);288-291 *
先天性巨结肠的遗传发病机制及前瞻性队列研究进展;张彦，等;《临床小儿外科杂志》;20180228;第17卷(第2期);90-93 *
精准医学在儿外科出生缺陷疾病谱中的应用;张彦，等;《临床小儿外科杂志》;20170831;第16卷(第4期);324-327、331 *

Also Published As

Publication number Publication date
CN113075410A (en) 2021-07-06

Similar Documents

Publication Publication Date Title
CN113092779B (en) Application of anti-NMDAR autoantibody as diagnosis marker of congenital megacolon
US7906291B2 (en) Method for diagnosing multiple sclerosis
CN113075410B (en) Application of anti-nRNP/Sm antibody as diagnosis marker of congenital megacolon
CN113075400B (en) Application of anti-double-stranded DNA antibody as diagnosis marker of Hirschmannin
JP2010019849A (en) Cvd assay
CN113075399B (en) Application of anti-single-chain DNA antibody as diagnosis marker of Hirschmannin
CN113092752B (en) Application of anti-SSA autoantibody as congenital megacolon diagnosis marker
JP6998626B2 (en) An immunological composition for diagnosing lung cancer using an autoantibody-antigen conjugate, a method for diagnosing lung cancer using the same, and a kit for diagnosing lung cancer containing the same.
KR20150140657A (en) Methods and compositions for diagnosing preeclampsia
Ziberna et al. A novel quantitative ELISA as accurate and reproducible tool to detect epidermal transglutaminase antibodies in patients with Dermatitis Herpetiformis
WO2022206585A1 (en) Antibody markers for diagnosing hirschsprung disease and application thereof
WO2016072464A1 (en) Arteriosclerosis and cancer detection method using deoxyhypusine synthase gene as indicator
US20230055382A1 (en) Detecting gut barrier dysfunction and/or cirrhosis
ES2889973T3 (en) Biosensor for male infertility
EP2812694A2 (en) Assays and methods for the diagnosis of ovarian cancer
CN113702464A (en) P-tau detection immunosensor and preparation and application methods thereof
JP2011209281A (en) Examination method and examination kit for urological cancer
JP5857385B2 (en) Bladder cancer diagnostic composition containing APE1 / REF-1 and bladder cancer diagnostic kit using the same
CN110687285A (en) Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus
WO2005031360A1 (en) Method of detecting or discriminating articular rheumatism and method of determining stage of disease or degree of dysfunction
CN114410767B (en) CMV and PRDM9 transposon fusion as congenital megacolon early diagnosis marker and application thereof
WO2024006460A9 (en) Peptide-based biomarkers and related aspects for disease detection
JP2023547188A (en) Proteins for the detection of schistosomiasis infections
WO2023030561A1 (en) Kit and method for simultaneous electrochemical determination of at least two protein analytes
CN116539894A (en) Application of CAPG in preparation of pathological myocardial hypertrophy detection reagent

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant